Distinct Contributions of DNA Methylation and Histone Acetylation to the Genomic Occupancy of Transcription Factors

Downloaded from genome.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press Research Distinct contributions of DNA methylation and histone acetylation to the genomic occupancy of transcription factors Martin Cusack,1 Hamish W. King,2 Paolo Spingardi,1 Benedikt M. Kessler,3 Robert J. Klose,2 and Skirmantas Kriaucionis1 1Ludwig Institute for Cancer Research, University of Oxford, Oxford, OX3 7DQ, United Kingdom; 2Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom; 3Target Discovery Institute, University of Oxford, Oxford, OX3 7FZ, United Kingdom Epigenetic modifications on chromatin play important roles in regulating gene expression. Although chromatin states are often governed by multilayered structure, how individual pathways contribute to gene expression remains poorly understood. For example, DNA methylation is known to regulate transcription factor binding but also to recruit methyl-CpG binding proteins that affect chromatin structure through the activity of histone deacetylase complexes (HDACs). Both of these mechanisms can potentially affect gene expression, but the importance of each, and whether these activities are inte- grated to achieve appropriate gene regulation, remains largely unknown. To address this important question, we measured gene expression, chromatin accessibility, and transcription factor occupancy in wild-type or DNA methylation-deficient mouse embryonic stem cells following HDAC inhibition. We observe widespread increases in chromatin accessibility at retrotransposons when HDACs are inhibited, and this is magnified when cells also lack DNA methylation. A subset of these elements has elevated binding of the YY1 and GABPA transcription factors and increased expression. The pronounced additive effect of HDAC inhibition in DNA methylation–deficient cells demonstrates that DNA methylation and histone deacetylation act largely independently to suppress transcription factor binding and gene expression. [Supplemental material is available for this article.] The most abundant DNA modification in mammals is 5-methylcy- ylation is reduced or absent (Domcke et al. 2015; Maurano et al. tosine (5mC) which is found predominantly within CpG dinucle- 2015; Yin et al. 2017). otides (Lister et al. 2009; Stadler et al. 2011). DNA methylation is In addition to directly altering the binding of transcription deposited and maintained through the concerted activity of three factors, methylated CpG is recognized by methyl binding essential DNA methyltransferases (DNMT1, DNMT3A, DNMT3B) domain-containing (MBD) and zinc-finger methyl-cytosine bind- (Goll and Bestor 2005) and plays important roles in transcriptional ing proteins (Hendrich and Bird 1998; Lopes et al. 2008; Quenne- repression. For example, DNA methylation is known to regulate ville et al. 2011). MBD proteins interact with histone deacetylase imprinted genes (Kaneda et al. 2004), some tissue-restricted genes (HDAC) enzymes and promote the formation of transcriptionally (De Smet et al. 1999; Maatouk et al. 2006; Borgel et al. 2010), evo- repressive chromatin environments (Nan et al. 1998; Zhang et al. lutionarily young retrotransposons (Davis et al. 1989; Walsh et al. 1999; Kokura et al. 2001; Yoon et al. 2003) via the removal of acetyl 1998; Castro-Diaz et al. 2014), genes on the inactive X groups from lysine residues in histones. Chromosome (Beard et al. 1995; Hansen et al. 1996), and certain Despite the active recruitment of HDACs to methylated tumor suppressor genes in cancer cells (Toyota et al. 1999; Rhee CpGs, the extent to which DNA methylation relies on HDAC activ- et al. 2002; Robert et al. 2003). ity to affect gene expression is still poorly understood. HDAC inhi- Although 60%–80% of CpGs in the genome are methylated, bition, or disruption of the interaction between MBD proteins and DNA methylation is absent or reduced in regions bound by tran- the HDAC-containing complexes, has been shown to alleviate scription factors such as in CpG islands, gene promoters, and distal transcriptional silencing mediated by DNA methylation in report- regulatory elements (Stadler et al. 2011; Hon et al. 2013; Ziller et al. er gene assays (Jones et al. 1998; Nan et al. 1998; Ng et al. 1999; 2013). This observation led to the proposal that DNA methylation Lyst et al. 2013). Nevertheless, at other endogenous genes, inhib- may limit transcription factor occupancy. In support of this mod- iting HDACs did not recapitulate the effects on gene expression el, in vitro experiments have revealed that 5mC can affect the af- that manifest when DNA methylation is removed (Cameron finity of most DNA-binding proteins that contain CpG et al. 1999; Coffee et al. 1999; Brunmeir et al. 2010; Reichmann dinucleotides in their binding motif (Hu et al. 2013; Spruijt et al. et al. 2012). Therefore, although it is clear that DNA methylation 2013; Kribelbauer et al. 2017; Yin et al. 2017). Furthermore, certain is capable of altering TF-DNA interactions and recruiting HDACs, transcription factors (TFs), including NRF1 and CTCF, have been how these distinct mechanisms contribute to DNA methylation- shown to have altered binding profiles in cells where DNA meth- dependent repression at the genome scale remains unknown. Corresponding author: [email protected] Article published online before print. Article, supplemental material, and publi- © 2020 Cusack et al. This article, published in Genome Research, is available cation date are at http://www.genome.org/cgi/doi/10.1101/gr.257576.119. under a Creative Commons License (Attribution-NonCommercial 4.0 Interna- Freely available online through the Genome Research Open Access option. tional), as described at http://creativecommons.org/licenses/by-nc/4.0/. 30:1–14 Published by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/20; www.genome.org Genome Research 1 www.genome.org Downloaded from genome.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press Cusack et al. Failure to inactivate transcription and mobility of retrotrans- (Supplemental Fig. S1A) but yielded a robust increase in global his- posons has been linked to genome instability in cancer (Carreira tone acetylation as detected by immunoblotting (Fig. 1B). There et al. 2014; Tubio et al. 2014; Scott and Devine 2017; Schauer were no significant changes in global 5mC levels between TSA- et al. 2018) and the manifestation of neurological diseases and DMSO (solvent)-treated cells (Supplemental Fig. S1B). (Coufal et al. 2011). DNA methylation is an important contributor To examine histone acetylation in more detail, we performed to silencing of retrotransposons, where it acts in concert with his- native calibrated ChIP-seq for acetylated histone H3 (H3ac). TSA tone deacetylation and histone (H3K9) methylation (Bourc’his treatment led to a global increase in H3ac in wild-type and and Bestor 2004; Karimi et al. 2011; Rowe et al. 2013; Sharif DNMT.TKO cells both at previously hyperacetylated sites (ChIP- et al. 2016). Whether chromatin mechanisms are redundant or col- seq peaks) and the remaining genomic space (Fig. 1C–E; Supple- laborative while acting on different classes of retrotransposons is mental Fig. S1C). The increase in histone acetylation at repetitive incompletely understood. regions and unannotated genomic loci following HDAC inhibi- In this study, we aimed to determine individual and collabo- tion was greater in DNMT.TKO compared to wild-type cells (Fig. rative roles of DNA methylation and HDAC activity. To this pur- 1E). This observation coincides with high DNA methylation levels pose, we disrupted both processes, one at a time or in in the aforementioned regions in WT cells, whereas hyperacety- combination, in mouse embryonic stem cells and measured the lated sites contain low median DNA methylation (Supplemental impact on chromatin accessibility, transcription factor occupancy, Fig. S1D). This additive effect of DNA methylation loss and and gene regulation. HDAC inhibition on histone acetylation in these regions could be explained by enhanced recruitment of histone acetyltransfer- ases (HATs), potentiated by DNA-binding proteins with specificity Results to unmodified CpGs (e.g., CxxC-domain proteins). Alternatively, the lack of DNA methylation may disrupt the recruitment of Disruption of HDAC activity and DNA methylation in mouse HDACs by MBD-domain proteins, resulting in a lower capacity embryonic stem cells for compensatory mechanisms to deacetylate histones or prevent To determine the contribution of DNA methylation and the activity histone acetylation. of HDAC enzymes on transcriptional regulation, we treated wild- In summary, HDAC inhibition using TSA induces histone type (J1) or Dnmt3a/Dnmt3b/Dnmt1 triple knockout (DNMT.TKO) acetylation broadly across the genome with no detectable change mouse embryonic stem cells (Tsumura et al. 2006) with the HDAC in DNA methylation, enabling us to evaluate the extent and the inhibitor trichostatin A (TSA) (Fig. 1A). The chosen dose of TSA mode by which these two mechanisms contribute to chromatin had minimal effects on the morphology or cell cycle of mESCs function. A C B DE Figure 1. Disruption of HDAC activity and DNA methylation in mouse embryonic stem cells. (A) Schematic of the experimental approach used in this study. (B) Immunoblot analysis of global histone H3 and H4 acetylation levels after 36-h TSA treatment in mESCs. Samples were derived from three bio- logical replicate experiments. (C) Representative snapshot of genomic

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